Inflammatory bowel disease or IBD includes Crohn's disease and Ulcerative Colitis and affects as many as one million Americans. IBD causes inflammation of the intestinal tract and particularly the intestinal wall, but Crohn's disease and Ulcerative Colitis differ in their location and depth of infection. Crohn's Disease was once considered a disease of only of the ileum, but is now recognized to affect any part of the digestive tract form the mouth to the anus. However, the ileum and the colon are the most commonly involved areas of the digestive tract with Crohn's disease. Meanwhile, Ulcerative Colitis is mainly considered a mucosal disease of the colon.
The clinical features of Crohn's Disease and Ulcerative Colitis demonstrate significant overlap including: abdominal pain, diarrhea, fever, and weight loss. These common symptoms suggest a commonality between the two diseases. One of the most prominent histological features observed in patients with these IBD diseases is infiltration of neutrophils, a type of white blood cell (granulocyte) that help the cell to kill and digest microorganisms it has engulfed by phagocytosis, into the inflamed mucosa at an early stage of inflammation. Disease activity is linked to an influx of neutrophils into the mucosa and the formation of crypt abscesses.
Interleukin-1 (IL-1; IL-1α and IL-1β) and interleukin-18 (IL-18; also known as interferon-γ inducing factor) are two key cytokines involved in the initiation and amplification of the inflammatory process of inflammatory bowel diseases such as Crohn's Disease and Ulcerative Colitis. IL-1 and IL-18 are central pro-inflamatory cytokines that function to stimulate the expression of genes associated with inflammation and autoimmune diseases. While monocytes, macrophages or monocytic cell lines are among the most studied cells in the known literature on the processing and release of IL-1 and IL-18, these cytokines are also expressed in various types of epithelial cells, including intestinal epithelial cells.
Cells exposed to IL-1 demonstrate a large expression of prostaglandin-E2 (PEG-2), platelet activation factor and nitric oxide (NO) due to the fact that IL-1 induces expression of cyclooxygenase type 2(COX-2), type 2 phospholipase A and inducible nitric oxide synthase (iNOS). IL-18 is a pivotal cytokine for the development of T-helper type I (Th1) lymphocyte responses. The most prominent activity of I1-18 is to induce interferon-γ (INFγ) by acting synergistically with IL-12. IL-18 also up-regulates the production of IL-1 and TNF-α.
It is known that increased gut mucosal secretion of IL-18 and IL-1β may predict an acute relapse of Crohn's Disease. It is also known that IL-18 and IL-1β positively correlates with the clinical severity of Ulcerative Colitis and Crohn's Disease. Serum IL-18 concentration is known to be significantly higher in patients with Crohn's disease than normal controls. Furthermore, animal studies have demonstrated that blocking IL-1 or IL-18 production and/or activity attenuates intestinal inflammation and tissue destruction due to IBD, see, Arnead, W. P., Cytokine Growth Factor Rev., 13:323 (2002); Scheinin, et. al., Clin. Exp. Immunol., 133:38 (2003); Lochner, et. al., Pathobiology 70:164 (2002); and Siegmund, B., Biochem. Pharmacol., 64:1 (2002).
The genes for IL-1 and IL-18 do not encode a typical signal peptide and, as a result, newly synthesized proIL-1 and prolIL-18 are known to accumulate within the cytoplasm of activated monocytes, macrophages and intestinal epithelial cells. Conversion of the pro-forms of IL-1 and IL-18 to their mature form requires proteolytic action of caspase-1, see, Cerretti, et. al., Science, 257:97 (1992) and Akita et. al., J. Biol. Chem., 272:26595 (1997). However, both caspase-1 dependent and caspase-1 independent IL-18 processing in epithelial cells have been reported, see, e.g. Lu et. al., J. Immunol. 165:1463 (2000); Sugawara, et. al., J. Immunol. 167:6568 (2001).
To achieve efficient IL-1 and IL-18 export from epithelial cells, such cells must encounter a secondary stimulus that specifically activates the posttranslational processing events. When a lipopolysaccharides (LPS) injection is followed with an Adenosine triphosphate (ATP) injection to the peritoneal cavity of mice, large quantities of cell-dissociated, mature IL-1β are generated. Likewise, it is known that cell-free IL-1 may be detected in plasma following LPS activation of human whole blood ex vivo, but cytokine levels are dramatically increased by co-administration of ATP. It is further known that cell death alone is insufficient to generate IL-1 or IL-18 posttranslational processing and release, maturations of these cytokines requires an active cellular response.
Accordingly, it has been revealed by several researchers that ATP initiates IL-1β/IL-18 posttranslational processing via ATP's activation of a P2X7 receptor, see, Ferrari, et. al. J. Immunol., 159:1451 (1997); Hogquist, et. al., Proc. Natl. Acac. Sci. USA, 88:8485; and Mehta, et. al., J. Biol. Chem., 276:3820. However, it appears that the P2X7 receptor is not an obligate element for IL-1β posttranslational processing and release since LPS-activated P2X7 receptor−/− macrophages do not release mature IL-1β in response to subsequent ATP stimulation, and in fact, only release mature IL-1β when treated with nigercin, a non-relevant physiological stimulus. Thus, it is likely that other ligand(s) with higher affinity to the P2X7 receptor or a different ligand receptor pathway initiates IL-1β/IL-18 posttranslational processing. Accordingly, no physiologically relevant effectors have been identified in the prior art that either promote or inhibit IL-1β/IL-18 posttranslational processing IL-18 posttranslational processing and release.
Defensins are endogenous antimicrobial peptides produced by white blood cells (neutrophils) and by cells lining the intestinal wall that aid in fighting bacterial infections (Paneth cells). Defensins also play a role in the inflammation of the intestines, commonly known as inflammatory bowel disease or IBD, including Crohn's Disease and Ulcertive Colitis. In fact, overproductions of defensins are characteristic of patients diagnosed with such diseases. Thus, increased local presences of antimicrobial defensin peptides are positively correlated with intestinal inflammation and damage in patients with Crohn's disease and Ulcerative Colitis.
Defensins comprise two classes: α-defensins (HNP1-3, HD-5, HD-6, Crp-3, Crp-4 and PG-1) and β-defensins (HBD-1, HBD-2 and HBD-3). As demonstrated in FIG. 1, defensin peptides contain six cysteine residues in a conserved spacing pattern. As further demonstrated in FIG. 1, the difference between α-defensins and β-defensins can be identified by the spacing and connectivity of their six cysteine residues. Defensins are amphiphilic in nature and have the ability to form voltage gated pores in phospholipid bilayers that allow defensins to perturb the membranes of susceptible microbial targets.
The isolation and purification of natural defensin peptides are well described in the scientific and patent literature. In particular, such methods are described in U.S. Pat. No. 5,242,902, as well as in U.S. Pat. Nos. 4,543,252; 4,659,692; and 4,705,777, the disclosures of which are incorporated herein by reference.
It is known that defensins are increased in the intestinal mucosa of patients with IBD, see, e.g. Fahlgren et. al. Clin. Exp. Immunol., 131:90 (2003). HNP-1-3 have been detected in surface entrocytes of mucosa with active IBD, but not in controls suggesting that HNP-1-3 have the opportunity to interact with the lamina propria and with intestinal epithelial cells following loss of epithelial barrier integrity in IBD. Paneth cell produced α-defensins HD-5 and HD-6 have been found to be over 6 have been found to be over expressed in Crohn's disease patients and in the colonic mucosa of IBD patients.
Accordingly, although first identified as antimicrobial peptides, research has suggested that defensins can interact with host immune cells, thereby playing an important role in both innate and adaptive immune responses against bacterial infection. Furthermore, because defensins can also influence the function of epithelial cells, T cells, dendridic cells and monocytes, the role of defensisn in IBD is controversial. Nonetheless, the consensus from published data indicates that the presence of neutrophil and Paneth cell-derived α-defensins are increased in both Crohn's disease and Ulcerative Colitis.